Interleukin (IL)-12 and IL-18 are key mediators promoting inflammatory reactions. Localized inflammatory responses are important mechanisms to limit pathogen infections and to promote wound healing. However, excessive and uncontrolled production of IL-12 and IL-18 and resulting inappropriate inflammation is linked to tissue destruction and the debilitating symptoms of the growing number of inflammatory diseases. Processing and release of IL-12 and IL-18 occurs in macrophages in response to formation of inflammasomes. Inflammasomes are activated by cytosolic pattern recognition receptors of the Nod-like receptor (NLR) family in response to pathogen or host derived stress signals. Activated NLRs recruit pro-caspase-1 via the essential adaptor protein ASC (PYCARD), resulting in caspase-1 activation and release of IL-12 and IL-18. The molecular mechanisms that control inflammasome formation and activation are poorly understood, but have a high potential to provide the basis for novel strategies to interfere with IL-12 and IL-18 release for the treatment of inflammatory diseases. Our central hypothesis for the proposed study is that inflammasomes represent inducible, specialized cytosolic structures in macrophages, where inflammasome components are specifically recruited to activate caspase-1. Our hypothesis is based on preliminary imaging results from inflammasomes in macrophages, and in this study we propose to address the mechanism of inducible inflammasome formation and link it to the IL-12 and IL-18 release mechanism. Our hypothesis is based on our preliminary findings showing that (1) inflammasomes require inducible redistribution of inflammasome components;(2) inflammasomes are inducible formed in the cytosol of macrophages;(3) inflammasomes contain characteristic marker proteins that directly link it to specialized cellular ultrastructures. We propose two specific aims: Specific aim #1 will establish inflammasomes as distinct structures in macrophages, while specific aim #2 will determine the impact on IL-12 and IL-18 maturation as a consequence of disrupting formation of these structures. At the completion of this study, we expect to provide the currently elusive mechanism by which inflammasomes assemble in response to infection and stress, which will open new avenues for inhibiting maturation of IL-12 and IL-18. This study will contribute to our long-term goal to contribute to a better understanding of molecular mechanisms of innate immune responses leading to inflammatory pathway activation, in order that improved therapies to treat inflammatory and infectious diseases can be developed. Public Health Relevance: Excessive production of IL-12 and IL-18 are directly responsible for the symptoms of a number of inflammatory diseases with destructive pathogenesis, including some of the most common diseases of industrialized nations, including arthritis, asthma, inflammatory bowel disease, ulcerative colitis, atherosclerosis, periodontitis, type 2 Diabetes, lung fibrosis, multiple sclerosis, Alzheimer's disease, or stroke. Currently there are no effective treatments available, causing patients life-long symptoms and a huge economical and financial impact on our social and medical systems. IL-12 and IL- 18 require specialized protein complexes, referred to as inflammasomes for release, but the mechanism by which inflammasomes assemble are not well understood, but are expected to provide the basis for the development of treatment options for patients suffering from inflammatory diseases. As a consequence, in this study we propose to address the mechanism of inflammasome formation, and to link inflammasomes to existing cellular ultra structures, which is expected to open new avenues for preventing uncontrolled release of IL-12 and IL-18.